Thrombin-inhibitory protein from assassin bugs

ABSTRACT

A novel thrombin-inhibitory protein from assassin bugs with a molecular weight of about 12,000 dalton and the N terminus Glu-Gly-Gly-Glu-Pro-Cys-Ala-Cys-Pro-His-Ala-Leu-His-Arg-Val-Cys-Gly-Ser-Asp is described. The protein is suitable for controlling diseases.

The present invention relates to a novel thrombin-inhibitory protein from assassin bugs and to a process for preparing it.

Thrombin inhibitors are important therapeutic substances used, for example, for the prophylaxis or treatment of thromboses or arterial reocclusions.

German Offenlegungsschrift DE 39 31 839 describes a thrombin inhibitor which has been isolated from the argasid tick Ornithodoros moubata. This protein has a molecular weight of about 15,000 dalton, an isoelectric point at pH 4-5 and the N-terminal amino-acid sequence SDYEFPPPKKXRPG.

European Published Application EP 345 614 describes the thrombin inhibitor amblyommin which is isolated from bont ticks. This is a protein with a molecular weight of 20,000-30,000 dalton and an isoelectric point at pH 5.05-5.65.

However, to date no protein with thrombin-inhibitory action has yet been found to be suitable and advantageous as a drug in terms of high activity, lack of antigenicity, long biological half-life, and few side effects such as risk of hemorrhage.

It is an object of the present invention to provide novel thrombin inhibitors which are suitable as drugs in terms of the abovementioned properties.

We have found that this object is achieved by a novel thrombin-inhibitory protein isolated from assassin bugs.

The novel protein has the following physicochemical properties. Molecular sieve chromatography shows that it has a molecular weight of 20,000-24,000 dalton. A molecular weight of 12,000±2000 dalton is determined in an SDS polyacrylamide gel. The determination of the isoelectric point shows that it is at pH 3.7-4.7.

The protein binds specifically to a thrombin affinity column. It inhibits the biological activity of thrombin in an in vitro enzyme assay.

The following N-terminal amino-acid sequence was determined for the protein (SEQ ID NO: 1): Glu-Gly-Gly-Glu-Pro-Cys-Ala-Cys-Pro-His-Ala-Leu-His-Arg-Val-Cys-Gly-Ser-Asp

The protein according to the invention contains a sequence of 103 amino acids which is shown in sequence listing SEQ ID NO: 3.

The DNA coding for this sequence of 103 amino acids is likewise detailed in sequence listing SEQ ID NO: 2.

The protein according to the invention may additionally contain further amino acids at the C terminus. It is likewise possible for the protein to contain at the N terminus other amino-acid sequences such as natural or heterologous leader sequences or additional amino acids such as methionine.

It is possible to predict from the amino-acid sequence indicated in sequence listing SEQ ID NO: 3 that there are two domains in this molecule. The first domain is limited essentially by the cysteine residues at positions 6 and 48, and the second domain by the cysteine residues at positions 57 and 101.

The invention also relates to DNA sequences which code for proteins having a thrombin-inhibitory action and which are selected from the group formed by

a) DNA sequences with the structure described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, and

b) DNA sequences which hybridize under standard conditions with DNA sequences a).

These DNA sequences are for thrombin inhibitors having up to 6 domains.

The invention also relates to smaller proteins which contain only one of these two domains. Proteins according to the invention also include those which contain a plurality, preferably up to 10, of such domains.

In this connection, the minimum requirements for a single active domain are the amino acids (from SEQ ID NO: 7) from position 6 to 48, 57 to 101 or 119 to 160 or the amino acids from SEQ ID NO: 17 from position 6 to 48, 57 to 101, 120 to 161, 190 to 232, 241 to 285 or 303 to 344.

N-Terminal and C-terminal extensions do not interfere with the thrombin-inhibiting activity. Extensions of these types may contain sequences such as, for example, the amino acids 1 to 5, 49 to 56, 102 to 118, 161 to 170 from SEQ ID NO: 7 or 1 to 5, 49 to 56, 102 to 119, 162 to 189, 233 to 240, 286 to 302 or 345 to 354 from SEQ ID NO: 17.

These amino-acid sequences, which are distinguished by high flexibility and hydrophilicity, can be employed as spacers between the individual domains. Multidomain proteins of these types have a prolonged half-life without any essential reduction in the activity of the individual domains.

Owing to the insensitivity of the novel protein and its individual domains to extensions at the N terminus and C terminus, heterologous linkage of the novel thrombin inhibitor with other proteins is also possible. Examples of such proteins are tissue plasminogen activators, streptokinase, urokinase, antithrombin III and activated protein C. A protein which is important for lysis therapy is tPA, its muteins and derivatives. The advantage of such fusion proteins is an increased thrombous specificity with, at the same time, a reduction in the reocclusion rate.

For example, the amino-acid sequence of tissue plasminogen activator (EP 93619) at position 1-527 is extended with amino acids 1-104 of the double-headed thrombin inhibitor by attaching via suitable restriction sites the corrresponding nucleotide sequence from (SEQ ID NO: 2 downstream of the nucleotide sequence coding for tPA 1-157.

Conversely, however, it is also possible, for example, to extend the nucleotide sequence from amino acid 104 of the double-headed inhibitor with the sequence for tissue plasminogen activator by amino acid 1 of tPA becoming amino acid 105 of the heterotrimeric protein.

Such combination proteins result in a higher clot specificity because the thrombin inhibitor selectively binds to the thrombin entrapped in the thrombus and thus brings its fusion partner up to the clot.

An alternative strategy is to provide the fusion protein described above with a sequence which can be cleaved by proteases (e.g. a factor Xa cleavage site (ILE-GLU-GLY-ARG-X)) which results, after activation of the coagulation system, in liberation of the fusion partners in order to exert, for example, their fibrinolytic and anticoagulant effect. It is possible for a plurality, preferably from 2 to 10, fusion partners to be put together in this way.

Such proteins which contain a plurality of these domains are expediently prepared by genetic manipulation. For example, the DNA sequences which code for the domains are linked by conventional methods to give a synthetic multidomain gene, and this gene is expressed in a conventional manner.

The novel protein can be isolated from assassin bugs of the genus Rhodnius. To do this, the bugs are homogenized, expediently in a buffer at pH 6-9, preferably pH 7-8, with a homogenizer, preferably a mixer. The insoluble constituents are then removed, preferably by centrifugation.

The protein can be further purified by chromatographic methods, preferably ion exchange chromatography and/or affinity chromatography. A purification step by thrombin affinity chromatography is particularly preferred.

The purification of the protein can be monitored by a thrombin activity assay. It is expedient to use an optical assay in which a chromogenic substrate, for example Chromozym T, is converted by thrombin. The fractions containing the novel protein can be identified by their thrombin-inhibiting action on addition to this optical assay.

Genetic engineering methods are particularly suitable for preparing the protein according to the invention.

To do this, a cDNA gene bank from the assassin bug is constructed in a conventional manner. It is possible to isolate the gene coding for the protein according to the invention from this gene bank by, for example, preparing a DNA probe whose sequence is obtained from the N-terminal amino-acid sequence described above by translation back using the genetic code. The appropriate gene can be found and isolated by hybridization with this DNA probe.

However, it is also possible to employ the polymerase chain reaction (PCR) technique to prepare the appropriate gene. For example, a primer whose sequence has been obtained by translation back from the N-terminal amino-acid sequence described above, and a second primer whose sequence is complementary to the 3' end of the cDNA gene fragment, preferably with the sequence poly(dT), can be used to prepare the cDNA gene fragment for the protein according to the invention by the PCR technique. The appropriate gene can also be isolated by constructing an expression gene bank from assassin bugs and screening this with an antibody directed against the protein according to the invention.

Once the appropriate gene has been isolated, it can be expressed by genetic engineering methods in organisms, e.g. in bacteria, yeasts, eukaryotic cells, with the aid of an expression vector in a conventional manner.

It is preferable to use prokaryotes such as E. coli, and strongly expressing vectors, e.g. under the control of the inducible tac promoter as is present, for example, in the plasmid pMal-p2 (Protein Fusion and Purification System, "GeneExpress", New England Biolabs). This results in periplasmic expression of a fusion protein composed of the maltose binding protein and the thrombin inhibitor described. The fusion partner can be removed enzymatically after purification.

The general procedure for the preparation by genetic engineering of a novel protein when the partial amino-acid sequence is known is described in textbooks of genetic engineering, for example E. L. Winnacker, Gene und Klone, Verlag Chemie, Weinheim, 1984. The experimental conditions for the individual methods, such as construction of a gene bank, hybridization and expression of a gene, are described in T. Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory, 1990.

Standard conditions mean, for example, temperatures from 42° to 58° C. as an aqueous buffer solution with a concentration of from 0.1 to 1×SSC (1×SSC: 0.15M NaCl, 15 nM sodium citrate pH 7.2).

The protein according to the invention is preferably used in the form of its pharmaceutically acceptable salts.

The novel protein has anticoagulant properties. It can be used, for example, for the prophylaxis of thromboses or arterial reocclusions, for the treatment of thromboses, for conserving blood or in extracorporeal circulations.

The novel proteins are effective thrombin inhibitors. They can be used as drugs alone or together with known anticoagulant factors. The anticoagulant factors which are preferably employed are thrombin inhibitors, for example hirudin, factor Xa inhibitors, for example TAP (Waxman et al., Science 248 (1990) 593-596) or platelet aggregation inhibitors, for example kistrin (Dennis et al., Proc. Natl. Acad. Sci. USA 87 (1989) 2471-2475).

The invention is further illustrated by the following examples.

EXAMPLE 1

Purification of the thrombin-inhibitory protein from assassin bugs

A laboratory culture of the bugs (Rhodnius prolixus) was maintained at 28° C. and 80% relative humidity. The bugs were allowed to feed off rabbits at 30-day intervals. After the bugs had reached the last stage of development they were frozen at -20° C.

25 g of bugs were homogenized with 75 ml of 20 mM sodium phosphate buffer, 150 mM NaCl (pH 7.5). The homogenate was centrifuged at 20,000 rpm (Sorvall RC-3B, rotor SS-34) for 60 minutes. The precipitate was discarded.

The protein solution (supernatant) was loaded (60 ml/h) onto a Q-Sepharose® column (Pharmacia) which had been equilibrated in 20 mM sodium phosphate buffer pH 8.0 (diameter 2.5 cm, volume 50 ml).

The column was washed with 10 column volumes of equilibration buffer.

Then a linear gradient from 50 ml of 20 mM sodium phosphate (pH 8.0) to 50 ml of 20 mM sodium phosphate (pH 8.0), 1M NaCl was applied.

Active fractions (measured by thrombin inhibition) were collected.

The combined active fractions were loaded onto an affinity column with immobilized thrombin (diameter 1.5 cm, height 6.5 cm, volume 11.5 ml, 60 ml/h). The column was prepared as in Example 3.

The column was equilibrated with 20 mM sodium phosphate pH 7.5. After the protein solution had been loaded onto the column it was washed with 10 column volumes of equilibration buffer until the absorption at 280 nm decreased to zero.

It was then washed with 0.5M NaCl, 20 mM sodium phosphate buffer pH 7.5. This removed non-specifically adsorbed material.

Protein specifically bound to thrombin was eluted with 0.1M glycine, 0.5M NaCl pH 2.8. The column was then immediately readjusted to pH 7.5 with phosphate buffer.

The individual fractions were neutralized with 0.1M NaOH and examined for their inhibitory action on thrombin.

The fractions eluted by glycine/NaCl buffer pH 2.8 had a thrombin-inhibiting action.

The collected active fractions were, after neutralization, diluted with water (1:10) and loaded onto a Mono-Q® column (Pharmacia, volume 1 ml).

The column was equilibrated with 20 mM sodium phosphate buffer pH 7.5, 150 mM NaCl (buffer A). It was washed with buffer A until the absorption decreased to zero (10 minutes). The buffer was then changed over the course of 50 minutes to 20 mM sodium phosphate, pH 7.5, 800 mM NaCl (buffer B) (flow rate 0.5 ml/min).

Thrombin-inhibiting fractions were collected.

The collected fractions were further purified on an RP 318® (Biorad) HPLC column. The colum was equilibrated with 0.1% by weight of trifluoroacetic acid (TFA) in distilled water. The combined active fractions were loaded onto the column which was then eluted with a gradient to 0.1% by weight TFA, 100% acetonitrile at a flow rate of 1 ml/min over the course of one hour. The absorption was determined at 280 nm, and 0.5 ml fractions were collected. The collected fractions were concentrated to dryness and taken up in a phosphate-buffered saline (PBS) (0.8 g/l NaCl; 0.2 g/l HCl; 0.144 g/l sodium phosphate; 0.2 g/l potassium phosphate, pH 7.5), and the inhibitory activity was determined.

The protein was determined by the method of Bradford (Anal. Biochem., 72 (1976) (248-254) using bovine serum albumin (Boehringer Mannheim) as standard protein.

EXAMPLE 2

Determination of the inhibition of thrombin by the inhibitor

Thrombin (Boehringer Mannheim) was dissolved to a final concentration of 25 mU/ml in phosphate-buffered saline (PBS) (0.8 g/l NaCl, 0.2 g/l HCl, 0.144 g/l sodium phosphate, 0.2 g/l potassium phosphate, pH 7.5).

Chromozym TH (Boehringer Mannheim) was dissolved in 20 ml of H₂ O per vial.

50 μl of thrombin solution and 100 μl of Chromozym plus 25 μl of sample or buffer were placed in the wells of a microtiter plate. The absorption at 405 nm was measured at 37° C. immediately thereafter at time 0 and after 30 minutes.

When the sample was deeply colored another control without thrombin was treated as described above.

The activity of thrombin liberates a dye which absorbs at 405 nm from the chromogenic substrate. Inhibition of the thrombin by a thrombin inhibitor is evident from a smaller increase in the absorption at 405 nm and was quantified using a calibration plot.

EXAMPLE 3

Preparation of an affinity column with thrombin as ligand

a) Coupling:

2 g of CNBr-activated Sepharose (Pharmacia) were washed with 200 ml of 1 mM HCl on a suction funnel. The gel was taken up in 100 mM NaHCO₃, 500 mM NaCl pH 8.3 and immediately mixed with 10,000 units of thrombin (Sigma) in 100 mM NaHCO₃, 500 mM NaCl, pH 8.3.

The solution was gently shaken at 4° C. for 24 hours.

b) Blocking:

The gel material was allowed to settle and then washed with 100 mM NaHCO₃, 500 mM NaCl, pH 8.3. The Sepharose was then incubated with 100 mM NaHCO₃, 500 mM NaCl, 1M ethanolamine pH 8.3 for 2 hours.

c) Preparation:

To remove unbound thrombin, before use the gel material is washed once more in the column with 20 column volumes of PBS pH 7.4.

EXAMPLE 4

Determination of the molecular weight by molecular sieve chromatography

Material purified by Mono-Q® chromatography was passed at a flow rate of 1 ml/min in 20 mM sodium phosphate, 150 mM NaCl, pH 7.5 through a Spherogel® TSK 3000 SW molecular sieve column (Pharmacia, diameter 7.5 mm, height 60 cm).

The reference proteins were subjected to the same procedure (serum albumin MW 67,000 Da, ovalbumin MW 45,000 Da, chymotrypsinogen A MW 25,000 Da).

The logarithm of the molecular weight of the reference proteins was plotted against their elution time.

The thrombin inhibition by the eluted fractions of the sample was determined.

The logarithm of the molecular weight of the inhibitor was obtained from the intercept of the elution time on the calibration line.

The molecular weight was found to be 20,000-24,000 dalton by this determination.

EXAMPLE 5

Determination of the molecular weight by tricine SDS polyacrylamide gel electrophoresis

(Reference: Analytical Biochemistry, 166, (1987) 368-379 Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the range from 1-1000 kDa, Schagger, H. and von Jagow, G.)

The gel electrophoresis was carried out as stated in the reference at 20 mA and 1400 V, 30 watts.

The molecular weight determined by this method was 12,000±2000 dalton.

The reference proteins were intact myoglobin 17.2 kDa, myoglobin I+II 14.6 kDa, myoglobin I 8.2 kDa, myoglobin II 6.4 kDa, myoglobin III 2.6 kDa and myoglobin 1-14.

EXAMPLE 6

Determination of the sequence of the inhibitor Reduction and carboxymethylation

2.8 ml of protein solution (0.029 mg/ml) were mixed with 0.28 ml of buffer (1M tris/HCl, 0.5M guanidine hydrochloride, pH 8.6). Then 0.116 ml of dithiothreitol (DTT, 10 mg/ml) was added and the mixture was incubated at 37° C. for 10 minutes. After addition of 0.185 ml of iodoacetamide (10 mg/ml) the mixture was incubated at 37° C. for 90 minutes. The reaction was stopped with 0.073 ml of DTT as above.

The protein was purified by renewed reversed phase HPLC on RP 318®. The mixture was adjusted to a final concentration of 0.1% by weight trifluoroacetic acid (TFA) and separated in an HPLC system from Hewlett Packard (HP 1090 liquid chromatograph). The column was washed with solvent A (0.1% by weight TFA, 100% H₂ O) for 5 minutes. Then the proportion of solvent B (90% acetonitrile, 10% H₂ O, 0.1% by weight TFA) was increased to 50% over the course of 120 minutes. The absorption of the eluate at 214 and 280 run was measured. Absorbing fractions were collected. The protein was identified by SDS gel electrophoresis and subjected to sequence analysis in an Applied Biosystems 477 A protein sequencer in accordance with the manufacturer's instructions.

The following amino-terminal sequence was obtained (SEQ ID NO: 1):

    Glu-Gly-Gly-Glu-Pro-Cys-Ala-Cys-Pro-His-Ala-Leu-His-Arg-Val-Cys-Gly-Ser-Asp

EXAMPLE 7

Determination of the isoelectric point by isoelectric focusing

The determination was carried out with an LKB Multiphor 2117 (horizontal system) and an LKB 2103 power supply. Precast gels were employed (Pharmacia Ampholine PAGplate pH 3.5-9.5). The standard proteins employed were amyloglucosidase, pH 3.5; soybean trypsin inhibitor, pH 4.55; β-lactoglobulin A, pH 5.2; bovine carbonic anhydrase, pH 5.85; human carbonic anhydrase, pH 6.55; horse myoglobin, pH 6.85 and 7.35; lentil lectin, pH 8.15, 8.45, 8.65 and trypsinogen, pH 9.3.

Focusing conditions: 1500 volts, 30 watts. Buffers:

anode 1M phosphoric acid

cathode 1M sodium hydroxide solution

The plates were prefocused for 30 minutes to produce a pH gradient. The samples were loaded onto filter disks which lay on the gel. Focusing was continued for 30 minutes, the filter disks were removed and, after a further 30 minutes, focusing was stopped. The gels were immediately cut into 2 mm slices and transferred into distilled water. The protein eluted out of the gel slices overnight. The location of the thrombin inhibitor was determined by a thrombin inhibition assay. The pH can also be determined directly using a pH electrode. The reference substance hirudin had an isoelectric point at pH 3.5 and below. The novel inhibitor had an isoelectric point at pH 4.2±0.5.

EXAMPLE 8

Preparation of a DNA sequence which codes for a thrombin-inhibitory protein.

a) Isolation of RNA and preparation of a cDNA bank

Complete RNA was obtained from whole animals of the species Rhodnius prolixus by disruption in guanidinium thiocyanate. This was carried out using the materials and in accordance with the instructions for the RNA isolation kit supplied by Stratagene, La Jolla, Calif., USA (Catalog No. 200345). The polyadenylated messenger RNA was selected from the complete RNA by oligo(dT) affinity separation. This method was carried out with the materials and in accordance with the instructions for the PolyATract mRNA isolation system supplied by Promega, Madison, Wis., USA (Catalog No. Z5200).

The cDNA was synthesized from polyadenylated messenger RNA using materials and in accordance with the instructions of the ZAP-cDNA synthesis kit supplied by Stratagene, La Jolla, Calif., USA (Catalog No. 200400) and was then packaged in lambda phages using materials and in accordance with the instructions of the Uni-ZAP XR GigapackII Cloning Kit supplied by Stratagene, La Jolla, Calif., USA (Catalog No. 237611).

b) Preparation of oligonucleotide probes for the PCR

The starting point for the cloning of cDNA fragments by the polymerase chain reaction (PCR, see Molecular Cloning, 2nd edition (1989), Sambrook, J. et al., CSH Press, page 14.1 et seq.) was peptides with the amino-terminal protein sequence described in Example 6 (SEQ ID NO: 1).

On the basis of the genetic code it is possible to derive from the peptide sequence SEQ ID NO: 18:

    NH.sub.2 -Glu Gly Gly Glu Pro Cys Ala Cys Pro His Ala

(pos. 1-11) the nucleic acid sequence SEQ ID NO: 19:

    5'-GAA GGT GGT GAA CCN TGY GCN TGY CCN CAY GC-3'

of the coding DNA strand. The known degeneracy of the genetic code means that it is possible to employ a plurality of nucleotides (N: A, C, G, T; Y: C, T;) at some positions. This means that there is a complexity of 512 different oligonucleotides during the oligonucleotide synthesis.

The syntheses were carried out with an Applied Biosystems type 360A DNA synthesizer. The oligonucleotides were, after removal of the protective groups, purified by gel electrophoresis on an acrylamide/urea gel.

c) Preparation of DNA templates for the PCR

5 μg of complete RNA or 1 μg of poly(A)⁺ RNA from the RNA preparations detailed under a) were translated with the oligonucleotide A-B-T₁₈, (SEQ ID NO: 20):

    5'-CGAGGGGGATGGTCGACGGAAGCGACCTTTTTTTTTTTTTTTTT-3'

and with the aid of the enzyme reverse transcriptase into single-strand cDNA (1°cDNA). This was carried out with the materials and in accordance with the instructions for the SuperScript preamplification system supplied by Gibco BRL, Eggenstein, Germany (Catalog No.8089SA). After the reaction was complete, the low molecular weight constituents were removed on Biospin 30 columns supplied by BioRad, Richmond, Calif., USA (Catalog No. 732-6006).

d) PCR and cloning

The polymerase chain reaction was carried out in accordance with known protocols (see Molecular Cloning, 2nd edition (1989), Sambrook, J. et al., CSH Press, page 14.1 et seq.). A Perkin Elmer DNA thermal cycler was used for this, and a modification of the internal primer principle of Frohmann, M. A. et al. (Proc. Natl. Acad. Sci. USA 85 (1988) 8998-9002) was employed.

Specifically, the 1°cDNA from c) was amplified with the oligonucleotides SEQ ID NO: 19, see above, and SEQ ID NO: 21 (from A-B-T₁₈):

    5'-CGAGGGGGATGGTCGACGG-3'.

The PCR products were fractionated according to size by gel electrophoresis. Separated agarose slices with DNA fragments of increasing molecular weight were then employed in a second PCR with oligonucleotide SEQ ID NO: 19, see above, and SEQ ID NO: 22 (from A-B-T₁₈):

    5'-GATGGTCGACGGAAGCGACC-3'.

The PCR products selected in this way were likewise fractionated by gel electrophoresis and eluted by standard methods. After subcloning into the EcoRV cleavage site of the vector pBluescriptKS and replication of the plasmid in E. coli DH5alpha, sequence analysis of a clone with SEQ ID NO: 2 revealed an open reading frame of 103 amino acids with SEQ ID NO: 3 which contains the peptide described in SEQ ID NO: 1. The clone was then called pRPTI.

e) Screening of the cDNA bank

1×10⁶ recombinant phages of Rhodnius prolixus cDNA bank were subjected to screening with a probe corresponding to SEQ ID NO: 2. Known protocols were used for this (see "Molecular Cloning", 2nd edition (1989), Sambrook, J. et al., CSH Press). The positive clones underwent sequence analysis and are listed as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16.

f) Heterologous expression of the thrombin inhibitor

To prepare the recombinant thrombin inhibitor initially cDNA sequence SEQ ID NO: 2 coding for positions 1-103 of amino-acid sequence SEQ ID NO: 3 underwent PCR amplification by the methods described and were cloned with suitable ends in the XmnI and BamHI cleavage sites of the bacterial expression vector pMAL-p2 (Protein Fusion and Purification System, GeneExpress, New England Biolabs No. 800). This cloning leads to fusion of the bacterial maltose binding protein with the described thrombin inhibitor in the same reading frame. E. coli DH5alpha cells were transformed with the resulting plasmid, and the recombinant fusion protein was expressed and purified in accordance with the manufacturer's instructions. The thrombin inhibitor can be separated enzymatically from the fusion partner by utilizing a factor Xa cleavage site at the point of fusion. The yield of the thrombin inhibitor which has undergone periplasmic expression in E. coli is 6000 units/L of culture (standard: National Institutes of Health).

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 22                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       GluGlyGlyGluProCysAla CysProHisAlaLeuHisArgValCys                              151015                                                                         GlySerAsp                                                                      (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 350 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                        (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       GAAGGTGGTGAACCGTGCGCTTGCCCCCACGCTCTGCATAGAGTTTGC48                             GluGlyGlyGluProCysAlaCysProHisAlaLeuHisArgValCys                               15 1015                                                                        GGCTCTGATGGTGAAACTTATAGCAACCCTTGTACGCTGAACTGTGCT96                             GlySerAspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAla                               20 2530                                                                        AAATTCAATGGAAAGCCAGAACTTGTAAAAGTCCATGATGGTCCTTGC144                            LysPheAsnGlyLysProGluLeuValLysValHisAspGlyProCys                               35 4045                                                                        GAACCGGATGAGGATGAAGATGTTTGCCAAGAATGTGATGGTGATGAA192                            GluProAspGluAspGluAspValCysGlnGluCysAspGlyAspGlu                               50 5560                                                                        TACAAACCAGTTTGCGGATCTGACGACATAACTTACGATAACAACTGT240                            TyrLysProValCysGlySerAspAspIleThrTyrAspAsnAsnCys                               6570 7580                                                                      CGACTAGAGTGTGCCTCTATCTCTTCCAGCCCAGGAGTTGAACTGAAA288                            ArgLeuGluCysAlaSerIleSerSerSerProGlyValGluLeuLys                               85 9095                                                                        CATGAAGGACCTTGTAGAACCGAAAAAAAAAAAAAAAAAAAAGGTCGCTTC339                         HisGluGlyProCysArgThr                                                          100                                                                            CGTCGACCATC 350                                                                (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 103 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       GluGlyGlyGluProCysAlaCysProHisAlaLeuHisArg ValCys                              151015                                                                         GlySerAspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAla                               202530                                                                         Ly sPheAsnGlyLysProGluLeuValLysValHisAspGlyProCys                              354045                                                                         GluProAspGluAspGluAspValCysGlnGluCysAspGlyAspGlu                               50 5560                                                                        TyrLysProValCysGlySerAspAspIleThrTyrAspAsnAsnCys                               65707580                                                                       ArgLeuGluCysAlaSerIleSer SerSerProGlyValGluLeuLys                              859095                                                                         HisGluGlyProCysArgThr                                                          100                                                                            (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 447 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       AAACTTATAGCAACCCTTGTACGCTGAACTGTGCTAAACACAATGGA47                              ThrTyrSerAsnProCysThrLeuAsnCysAlaLysHisAs nGly                                 151015                                                                         AAGCCAGGTCTTGTAAAAGTCCATGATGGTCCTTGCGAACCGGATGAG95                             LysProGlyLeuValLysValHisAspGlyProCysGlu ProAspGlu                              202530                                                                         GATGAAGATGTTTGCCAAGAATGTGATGATGTCGATTACGAACCAGTT143                            AspGluAspValCysGlnGluCysAspAspValAsp TyrGluProVal                              354045                                                                         TGTGGAACTGACGACAAAACTTACGATAACAACTGTCGACTAGAGTGT191                            CysGlyThrAspAspLysThrTyrAspAsnAsnCys ArgLeuGluCys                              505560                                                                         GCCTCTATCTCTTCCAGCCCAGGACTTGAACTGAAGCACACAGGAAAA239                            AlaSerIleSerSerSerProGlyLeuGluLeuLysHis ThrGlyLys                              657075                                                                         TGTCTACCCCATTTGGATTTTCCCGACCCAGTTTAAAGCTTGCACATAACGGA292                       CysLeuProHisLeuAspPheProAspProVal                                              80 8590                                                                        AAATGCACTATAGCAGAGTTATATCACGGTTTATTGTAAAAAAAGATTATATGAATTTAT352                CATAATATCAATAAAATAGCTTATTTTAAAAATATTGAACCAATTTAAATTTTCAACATA412                TGTATATGTAAAT AAATTTAAAAAAAAAAAAAAAA447                                        (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 90 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       ThrTyrSerAsnProCysThrLeuAsn CysAlaLysHisAsnGlyLys                              151015                                                                         ProGlyLeuValLysValHisAspGlyProCysGluProAspGluAsp                               2025 30                                                                        GluAspValCysGlnGluCysAspAspValAspTyrGluProValCys                               354045                                                                         GlyThrAspAspLysThrTyrAspAsnAsnCysArgLeuGluCys Ala                              505560                                                                         SerIleSerSerSerProGlyLeuGluLeuLysHisThrGlyLysCys                               65707580                                                                       LeuProHi sLeuAspPheProAspProVal                                                8590                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 732 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       AGCG TCTACTGTTGTTACTCGGATTGGCTGCACTCGTTGCAGCTGAA47                             ArgLeuLeuLeuLeuLeuGlyLeuAlaAlaLeuValAlaAlaGlu                                  -14-10-51                                                                      GGGGGGGAACCATGCGCATGTCCACATGCTCTGCATAGAGTTTGCGGC95                             GlyGlyGluProCysAlaCysProHisAlaLeuHisArgValCysGly                               51015                                                                          TCTGATGGTGAAACTTATAGCAACCCTTGTACGCTGAACTGTGCTAAA143                            SerAspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAlaLys                               202530                                                                          TTCAATGGCAAGCCAGAACTTGTAAAAGTCCATGATGGTCCTTGCGAA191                           PheAsnGlyLysProGluLeuValLysValHisAspGlyProCysGlu                               354045                                                                         CCGGAT GAGGATGAAGATGTTTGCCAAGAATGTGATGGTGATGAATAC239                           ProAspGluAspGluAspValCysGlnGluCysAspGlyAspGluTyr                               50556065                                                                        AAACCAGTTTGCGGATCTGACGGCATAACTTACGATAACAACTGTCGA287                           LysProValCysGlySerAspGlyIleThrTyrAspAsnAsnCysArg                               707580                                                                         CTAGAGTGTGCCTCTATCTCTTCCAGCCCAGGAGTTGAACTGAAACAT335                            LeuGluCysAlaSerIleSerSerSerProGlyValGluLeuLysHis                               859095                                                                         GAAGGGATATGTAGAAAGGAGGAAAAGAAACTTCCTAAAAGATCTGTG383                            GluGlyIleCysArgLysGluGluLysLysLeuProLysArgSerVal                               100105110                                                                       GGATTGGAACATACATGCGTCTGTCCTTATAATTATTTCCCGGTTTGC431                           GlyLeuGluHisThrCysValCysProTyrAsnTyrPheProValCys                               115120125                                                                      GGAACA GATGGGGAAACCTATCCCAACTTGTGCGCCCTCCAATGTCGT479                           GlyThrAspGlyGluThrTyrProAsnLeuCysAlaLeuGlnCysArg                               130135140145                                                                    ATGAGAGAAGTTCCAGGACTTGAACTGAAGCACACAGGAAAATGTCTA527                           MetArgGluValProGlyLeuGluLeuLysHisThrGlyLysCysLeu                               150155160                                                                      CCCCATTTGGATTTTCCCGACCCAGTTTAAAGCTTGCACATAACGGA574                             ProHisLeuAspPheProAspProVal                                                    165170                                                                         AAATGCACTATAGCAGAGTTATATCACGGTTTATTGTAAA AAAAGATTATATGAATTTAT634               CATAATATCAATAAAATAGCTTATTTTAAAAATATTGAACCAATTTAAATTTTCAACATA694                TGTATATGTAAATAAATTTAAAAAAAAAAAAAAAAAAA732                                      (2) INFORMATION FOR SEQ ID NO: 7:                                               (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 184 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       ArgLeuLeuLeuLeuLeuGlyLeuAlaAlaLeuValAlaAlaGluGly                               -14-10-5 1                                                                     GlyGluProCysAlaCysProHisAlaLeuHisArgValCysGlySer                               51015                                                                          AspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAlaLys Phe                              202530                                                                         AsnGlyLysProGluLeuValLysValHisAspGlyProCysGluPro                               35404550                                                                       AspGluAs pGluAspValCysGlnGluCysAspGlyAspGluTyrLys                              556065                                                                         ProValCysGlySerAspGlyIleThrTyrAspAsnAsnCysArgLeu                               70 7580                                                                        GluCysAlaSerIleSerSerSerProGlyValGluLeuLysHisGlu                               859095                                                                         GlyIleCysArgLysGluGluLysLys LeuProLysArgSerValGly                              100105110                                                                      LeuGluHisThrCysValCysProTyrAsnTyrPheProValCysGly                               115120125 130                                                                  ThrAspGlyGluThrTyrProAsnLeuCysAlaLeuGlnCysArgMet                               135140145                                                                      ArgGluValProGlyLeuGluLeuLysHisThrGlyLysCys LeuPro                              150155160                                                                      HisLeuAspPheProAspProVal                                                       165170                                                                         (2) INFORMATION FOR SEQ ID NO: 8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 677 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       ATGAAGCGTCTACTGTTGTTACTCGGATTGGCTGCACTCGTTGCAGCT48                             MetLysArgLeuLeuLeuLeuLeuGlyLeuAlaAlaLeuValAlaAla                                -15-10-5                                                                      GAAGGGGGGGAACCATGCGCATGTCCACATGCTCTGCATAGAGTTTGC96                             GluGlyGlyGluProCysAlaCysProHisAlaLeuHisArgValCys                               1 51015                                                                        GGCTCTGATGGTGAAACTTATAGCAACCCTTGTACGCTGAACTGTGCT144                            GlySerAspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAla                                202530                                                                        AAATTTAATGGAAAGCCAGAACTTGTAAAAGTCCATGATGGTCCTTGC192                            LysPheAsnGlyLysProGluLeuValLysValHisAspGlyProCys                                354045                                                                        GAACCGGATGAGGATGAAGATGTTTGCCAAGAATGTGATGGTGATGAA240                            GluProAspGluAspGluAspValCysGlnGluCysAspGlyAspGlu                                505560                                                                        TACAAACCAGTTTGCGGATCTGACGGCATAACTTACGATAACAACTGT288                            TyrLysProValCysGlySerAspGlyIleThrTyrAspAsnAsnCys                               65 707580                                                                      CGACTAGAGTGTGCCTCTATCTCTTCCAGCCCAGGAGTTGAACTGAAA336                            ArgLeuGluCysAlaSerIleSerSerSerProGlyValGluLeuLys                                859095                                                                        CATGAAGGGATATGTAGAAAGGAGGAAAAGAAACTTCCTAAAAGATCT384                            HisGluGlyIleCysArgLysGluGluLysLysLeuProLysArgSer                                100105110                                                                     GTGGGATTGGAACATACATGCGTCTGTCCTTATAATTATTTCCCGGTT432                            ValGlyLeuGluHisThrCysValCysProTyrAsnTyrPheProVal                                115120125                                                                     TGCGGAACAGATGGGGAAACCTATCCCAACTTGTGCGCCCTCCAATGT480                            CysGlyThrAspGlyGluThrTyrProAsnLeuCysAlaLeuGlnCys                               1 30135140                                                                     CGTATGAGAGAAGTTCCAGGACTTGAACTGAAGCACACAGGAAAATGT528                            ArgMetArgGluValProGlyLeuGluLeuLysHisThrGlyLysCys                               145 150155160                                                                  CTACCCCATTTGGATTTTCCCGACCCAGTTTAAAGCTTGCACATAACGGA578                          LeuProHisLeuAspPheProAspProVal                                                 165 170                                                                        AAATGCACTATAGCAGAGTTATATCACGGTTTATTGTAAAAAAAGGATTATATGAATTTA638                TCATAATATCAATAAAATAGCTTATTTTAAAAATATTGA677                                     (2) INFORMATION FOR SEQ ID NO: 9:                                              (i) SEQUENCE CHARACTERISTICS:                                                   (A) LENGTH: 186 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                       MetLysArgLeuLeuLeuLeuLeuGlyLeuAlaAlaLeuValAlaAla                               -15-10-5                                                                       GluGlyGly GluProCysAlaCysProHisAlaLeuHisArgValCys                              151015                                                                         GlySerAspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAla                               20 2530                                                                        LysPheAsnGlyLysProGluLeuValLysValHisAspGlyProCys                               354045                                                                         GluProAspGluAspGluAspValCys GlnGluCysAspGlyAspGlu                              505560                                                                         TyrLysProValCysGlySerAspGlyIleThrTyrAspAsnAsnCys                               657075 80                                                                      ArgLeuGluCysAlaSerIleSerSerSerProGlyValGluLeuLys                               859095                                                                         HisGluGlyIleCysArgLysGluGluLysLysLeuProLysA rgSer                              100105110                                                                      ValGlyLeuGluHisThrCysValCysProTyrAsnTyrPheProVal                               115120125                                                                      CysGlyThr AspGlyGluThrTyrProAsnLeuCysAlaLeuGlnCys                              130135140                                                                      ArgMetArgGluValProGlyLeuGluLeuLysHisThrGlyLysCys                               145150 155160                                                                  LeuProHisLeuAspPheProAspProVal                                                 165170                                                                         (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 566 base pairs                                                     (B) TYPE: nucleic acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      AAAAGTCCATGATGGTCCTTGCGAACCGGATGAGGATGAAGATGTT46                               LysValHisAspGlyProCysGluProAspGluAspGluAspVal                                  1 51015                                                                        TGCCAAGAATGTGATGATGTCGATTACGAACCAGTTTGTGGAACTGAC94                             CysGlnGluCysAspAspValAspTyrGluProValCysGlyThrAsp                                202530                                                                        GACAAAACTTACGATAACAACTGTCGACTAGAGTGTGCCTCTATCTCT142                            AspLysThrTyrAspAsnAsnCysArgLeuGluCysAlaSerIleSer                                354045                                                                        TCCAGCCCAGGAGTTGAACTGAAACATGAAGGGATATGTAGAAAGGAG190                            SerSerProGlyValGluLeuLysHisGluGlyIleCysArgLysGlu                                505560                                                                        GAAAAGAAACTTCCTAAAAGATCTGTGGGATTGGAACATACATGCGTC238                            GluLysLysLeuProLysArgSerValGlyLeuGluHisThrCysVal                               65 7075                                                                        TGTCCTTATAATTATTTCCCGGTTTGCGGAACAGATGGGGAAACCTAT286                            CysProTyrAsnTyrPheProValCysGlyThrAspGlyGluThrTyr                               80 859095                                                                      CCCAACTTGTGCGCCCTCCAATGTCGTATGAGAGAAGTTCCAGGACTT334                            ProAsnLeuCysAlaLeuGlnCysArgMetArgGluValProGlyLeu                                100105110                                                                     GAACTGAAGCACACAGGAAAATGTCTACCCCATTTGGATTTTCCCGAC382                            GluLeuLysHisThrGlyLysCysLeuProHisLeuAspPheProAsp                                115120125                                                                     CCAGTTTAAAGCTTGCACATAACGGAAAATGCACTATAGCAGAGGTATATCACGGT438                    ProVal                                                                         130                                                                            TTATTGTAAAAAAAGGTTATATGAATTTATCATAATATCAATT AAAATAGCTTATTTTAA498               AAATATTGCCCATTTAAATTTTCAACATATGTATATGTAAATAAATTTAAAAAAAAAAAA558                AAAAAAAA566                                                                    (2) INFORMATION FOR SEQ ID NO: 11:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                      LysValHisAspGlyProCysGluProAspGluAspGluAspValCys                               1510 15                                                                        GlnGluCysAspAspValAspTyrGluProValCysGlyThrAspAsp                               202530                                                                         LysThrTyrAspAsnAsnCysArgLeuGluCysAlaSerIleSerSe r                              354045                                                                         SerProGlyValGluLeuLysHisGluGlyIleCysArgLysGluGlu                               505560                                                                         LysLysLeuProLysArg SerValGlyLeuGluHisThrCysValCys                              65707580                                                                       ProTyrAsnTyrPheProValCysGlyThrAspGlyGluThrTyrPro                               85 9095                                                                        AsnLeuCysAlaLeuGlnCysArgMetArgGluValProGlyLeuGlu                               100105110                                                                      LeuLysHisThrGlyLysCysLeuProH isLeuAspPheProAspPro                              115120125                                                                      Val                                                                            (2) INFORMATION FOR SEQ ID NO: 12:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 326 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (x i) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GTCGATTACGAACCAGTTTGTGGAACTGACGACAAAACTTACGATAAC48                             ValAspTyrGluProValCysGlyThrAspAspLysThrTyrAspAsn                               1510 15                                                                        AACTGTCGACTAGAGTGTGCCTCTATCTCTTCCAGCCCAGGAGTTGAA96                             AsnCysArgLeuGluCysAlaSerIleSerSerSerProGlyValGlu                               2025 30                                                                        CTGAAACATGAAGGGATATGTAGAAAGGAGGAAAAGAAACTTCCTAAA144                            LeuLysHisGluGlyIleCysArgLysGluGluLysLysLeuProLys                               3540 45                                                                        AGATCTGTGGGATTGGAACATACATGCGTCTGTCCTTATAATTATTTC192                            ArgSerValGlyLeuGluHisThrCysValCysProTyrAsnTyrPhe                               505560                                                                         CCGGTTTGCGGAACAGATGGGGAAACCTATCCCAACTTGTGCGGCCTC240                            ProValCysGlyThrAspGlyGluThrTyrProAsnLeuCysGlyLeu                               657075 80                                                                      CAATGTCGTATGAGAGAAGTTCCAGGACTTGAACTGAAGCACACAGGA288                            GlnCysArgMetArgGluValProGlyLeuGluLeuLysHisThrGly                               8590 95                                                                        AAATGTCTACCCCATTTGGATTTTCCCGACCCAGTTTA326                                      LysCysLeuProHisLeuAspPheProAspProVal                                           100105                                                                         (2) INFORMATION FOR SEQ ID NO: 13:                                             ( i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                      ValAspTyrGluProValCysGlyThrAspAspLysThrTyrAspAsn                               1510 15                                                                        AsnCysArgLeuGluCysAlaSerIleSerSerSerProGlyValGlu                               202530                                                                         LeuLysHisGluGlyIleCysArgLysGluGluLysLysLeuProLys                               354045                                                                         ArgSerValGlyLeuGluHisThrCysValCysProTyrAsnTyrPhe                               505560                                                                         ProValCysGlyThrAspG lyGluThrTyrProAsnLeuCysGlyLeu                              65707580                                                                       GlnCysArgMetArgGluValProGlyLeuGluLeuLysHisThrGly                               85 9095                                                                        LysCysLeuProHisLeuAspPheProAspProVal                                           100105                                                                         (2) INFORMATION FOR SEQ ID NO: 14:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 616 base pairs                                                     (B) TYPE: nucleic acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                      GCGTCTACTGTTGTTACTCGGATTGGCTGCACTCGTTGCAGCTGAA46                               ArgLeuLeuLeuLeuLeuGlyLeuAlaAlaLeuValAlaAlaGlu                                  -14 -10-51                                                                     GGGGGGGAACCATGCGCATGTCCACATGCTCTGCATAGAGTTTGCGGC94                             GlyGlyGluProCysAlaCysProHisAlaLeuHisArgValCysGly                                51015                                                                         TCTGATGGTGAAACTTATAGCAACCCTTGTACGCTGAACTGTGCTAAA142                            SerAspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAlaLys                                202530                                                                        TTCAATGGAAAGCCAGAACTTGTAAAAGTCCATGATGGTCCTTGCGAA190                            PheAsnGlyLysProGluLeuValLysValHisAspGlyProCysGlu                               35 4045                                                                        CCGGATGAGGATGAAGATGTTTGCCAAGAATGTGATGATGTCGATTAC238                            ProAspGluAspGluAspValCysGlnGluCysAspAspValAspTyr                               50 556065                                                                      GAACCAGTTTGTGGAACTGACGACAAAACTTACGATAACAACTGTCGA286                            GluProValCysGlyThrAspAspLysThrTyrAspAsnAsnCysArg                                707580                                                                        CTAGAGTGTGCCTCTATCTCTTCCAGCCCAGGAGTTGAACTGAAACAT334                            LeuGluCysAlaSerIleSerSerSerProGlyValGluLeuLysHis                                859095                                                                        GAAGGGATATGTAGAAAGGAGGAAAAGAAACTTCCTAAAAGATCTGTG382                            GluGlyIleCysArgLysGluGluLysLysLeuProLysArgSerVal                                100105110                                                                     GGATTGGAACATACATGCGTCTGTCCTTATAATTATTTCCCGGTTTGC430                            GlyLeuGluHisThrCysValCysProTyrAsnTyrPheProValCys                               115 120125                                                                     GGAACAGATGGGGAAACCTATCCCAACTTGTGCGCCCTCCAATGTCGT478                            GlyThrAspGlyGluThrTyrProAsnLeuCysAlaLeuGlnCysArg                               130 135140145                                                                  ATGAGAGAAGTTCCAGGACTTGAACTGAAGCACACAGGAAAATGTCTA526                            MetArgGluValProGlyLeuGluLeuLysHisThrGlyLysCysLeu                                150155160                                                                     CCCCATTTGGATTTTCCCGACCCAGTTTAAAGCTTGCACATAACGGA573                             ProHisLeuAspPheProAspProVal                                                    1651 70                                                                        AAATGCACTATAGCAGAGTTATATCACGGTTTATTGTAAAAAA616                                 (2) INFORMATION FOR SEQ ID NO: 15:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 184 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                      ArgLeuLeuLeu LeuLeuGlyLeuAlaAlaLeuValAlaAlaGluGly                              -14-10-51                                                                      GlyGluProCysAlaCysProHisAlaLeuHisArgValCysGlySer                               5 1015                                                                         AspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAlaLysPhe                               202530                                                                         AsnGlyLysProGluLeuValLysValHisAspG lyProCysGluPro                              35404550                                                                       AspGluAspGluAspValCysGlnGluCysAspAspValAspTyrGlu                               5560 65                                                                        ProValCysGlyThrAspAspLysThrTyrAspAsnAsnCysArgLeu                               707580                                                                         GluCysAlaSerIleSerSerSerProGlyValGluLeuLysHi sGlu                              859095                                                                         GlyIleCysArgLysGluGluLysLysLeuProLysArgSerValGly                               100105110                                                                      LeuGluHisThrCys ValCysProTyrAsnTyrPheProValCysGly                              115120125130                                                                   ThrAspGlyGluThrTyrProAsnLeuCysAlaLeuGlnCysArgMet                               135 140145                                                                     ArgGluValProGlyLeuGluLeuLysHisThrGlyLysCysLeuPro                               150155160                                                                      HisLeuAspPheProAspProVal                                                        165170                                                                        (2) INFORMATION FOR SEQ ID NO: 16:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1282 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                      AGCGTCTACTGTTGTTACTCGGATTGGCTGCA CTCGTTGCAGCTGAA47                             ArgLeuLeuLeuLeuLeuGlyLeuAlaAlaLeuValAlaAlaGlu                                  -14-10-51                                                                      GGGGGGGAACCATGCGCATGTCCACA TGCTCTGCATAGAGTTTGCGGC95                            GlyGlyGluProCysAlaCysProHisAlaLeuHisArgValCysGly                               51015                                                                          TCTGATGGTGAAACTTATAGCAACCC TTGTACGCTGAACTGTGCTAAA143                           SerAspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAlaLys                               202530                                                                         TTCAATGGAAAGCCAGAACTTGTAAAAGT CCATGATGGTCCTTGCGAA191                           PheAsnGlyLysProGluLeuValLysValHisAspGlyProCysGlu                               354045                                                                         CCGGATGAGGATGAAGATGTTTGCCAAGAATGTGA TGGTGATGAATAC239                           ProAspGluAspGluAspValCysGlnGluCysAspGlyAspGluTyr                               50556065                                                                       AAACCAGTTTGCGGATCTGACGACATAAC TTACGATAACAACTGTCGA287                           LysProValCysGlySerAspAspIleThrTyrAspAsnAsnCysArg                               707580                                                                         CTAGAGTGTGCCTCTATCTCTTCCAG CCCAGGAGTTGAACTGAAACAT335                           LeuGluCysAlaSerIleSerSerSerProGlyValGluLeuLysHis                               859095                                                                         GAAGGACCTTGTAGAACCGAGGAAAA GAAAATTCTTAAAAGATCTGAT383                           GluGlyProCysArgThrGluGluLysLysIleLeuLysArgSerAsp                               100105110                                                                      GAATTCGAAATGTATAGATGCGCATGTCC GAAAATATATTATCCGGTT431                           GluPheGluMetTyrArgCysAlaCysProLysIleTyrTyrProVal                               115120125                                                                      TGCGGAACAGATGGTGAAACCTATCCCAACTTGTG CGTCCTCGAATGT479                           CysGlyThrAspGlyGluThrTyrProAsnLeuCysValLeuGluCys                               130135140145                                                                   CATATGAGAATGAATCCAGGACTTCAATT GCACCATTATGGACATTGT527                           HisMetArgMetAsnProGlyLeuGlnLeuHisHisTyrGlyHisCys                               150155160                                                                      CAACATCATCATCACCATCATCCTCC TCCTCATCACCATCATCATCAT575                           GlnHisHisHisHisHisHisProProProHisHisHisHisHisHis                               165170175                                                                      CATCCTCATCACACCACTGAGAAACC AGTAGAACCATGCGCATGTCCA623                           HisProHisHisThrThrGluLysProValGluProCysAlaCysPro                               180185190                                                                      CATGCTCTGCATAGAGTTTGCGGCTCTGA TGGTGAAACTTATAGCAAC671                           HisAlaLeuHisArgValCysGlySerAspGlyGluThrTyrSerAsn                               195200205                                                                      CCTTGTACGCTGAACTGTGCTAAACACAATGGAAA GCCAGGTCTTGTA719                           ProCysThrLeuAsnCysAlaLysHisAsnGlyLysProGlyLeuVal                               210215220225                                                                   AAAGTCCATGATGGTCCTTGCGAACCGGA TGAGGATGAAGATGTTTGC767                           LysValHisAspGlyProCysGluProAspGluAspGluAspValCys                               230235240                                                                      CAAGAATGTGATGATGTCGATTACGA ACCAGTTTGTGGAACTGACGAC815                           GlnGluCysAspAspValAspTyrGluProValCysGlyThrAspAsp                               245250255                                                                      AAAACTTACGATAACAACTGTCGACT AGAGTGTGCCTCTATCTCTTCC863                           LysThrTyrAspAsnAsnCysArgLeuGluCysAlaSerIleSerSer                               260265270                                                                      AGCCCAGGAGTTGAACTGAAACATGAAGG GATATGTAGAAAGGAGGAA911                           SerProGlyValGluLeuLysHisGluGlyIleCysArgLysGluGlu                               275280285                                                                      AAGAAACTTCCTAAAAGATCTGTGGGATTGGAACA TACATGCGTCTGT959                           LysLysLeuProLysArgSerValGlyLeuGluHisThrCysValCys                               290295300305                                                                   CCTTATAATTATTTCCCGGTTTGCGGAAC AGATGGGGAAACCTATCCC1007                          ProTyrAsnTyrPheProValCysGlyThrAspGlyGluThrTyrPro                               310315320                                                                      AACTTGTGCGCCCTCCAATGCCGTAT GAGAGAAGTTCCAGGACTTGAA1055                          AsnLeuCysAlaLeuGlnCysArgMetArgGluValProGlyLeuGlu                               325330335                                                                      CTGAAGCACACAGGAAAATGTCTACC CCATTTGGATTTTCCCGACCCA1103                          LeuLysHisThrGlyLysCysLeuProHisLeuAspPheProAspPro                               340345350                                                                      GTTTAAAGCTTGCACATAACGGAAAATGCACTAT AGCAGAGTTATATCACGGT1156                     Val                                                                            TTATTGTAAAAAAAGATTATATGAATTTATCATAATATCAATAAAATAGCTTATTTTAAA1216               AATATTGAACCAATTTAAATTTTCAACATATGTATATGTAAATAAATTTAAAAAAAAAAA1276               AAAA AA1282                                                                    (2) INFORMATION FOR SEQ ID NO: 17:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 368 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                      ArgLeuLeuLeuLeuLeuG lyLeuAlaAlaLeuValAlaAlaGluGly                              -14-10-51                                                                      GlyGluProCysAlaCysProHisAlaLeuHisArgValCysGlySer                               5 1015                                                                         AspGlyGluThrTyrSerAsnProCysThrLeuAsnCysAlaLysPhe                               202530                                                                         AsnGlyLysProGluLeuValLysValHisAspGlyProCys GluPro                              35404550                                                                       AspGluAspGluAspValCysGlnGluCysAspGlyAspGluTyrLys                               5560 65                                                                        ProValCysGlySerAspAspIleThrTyrAspAsnAsnCysArgLeu                               707580                                                                         GluCysAlaSerIleSerSerSerProGlyValGluLeuLysHisGlu                                859095                                                                        GlyProCysArgThrGluGluLysLysIleLeuLysArgSerAspGlu                               100105110                                                                      PheGluMetTyrArgCysAlaCy sProLysIleTyrTyrProValCys                              115120125130                                                                   GlyThrAspGlyGluThrTyrProAsnLeuCysValLeuGluCysHis                               135 140145                                                                     MetArgMetAsnProGlyLeuGlnLeuHisHisTyrGlyHisCysGln                               150155160                                                                      HisHisHisHisHisHisProProProHisHis HisHisHisHisHis                              165170175                                                                      ProHisHisThrThrGluLysProValGluProCysAlaCysProHis                               180185190                                                                      AlaL euHisArgValCysGlySerAspGlyGluThrTyrSerAsnPro                              195200205210                                                                   CysThrLeuAsnCysAlaLysHisAsnGlyLysProGlyLeuValLys                                215220225                                                                     ValHisAspGlyProCysGluProAspGluAspGluAspValCysGln                               230235240                                                                      GluCysAspAspVa lAspTyrGluProValCysGlyThrAspAspLys                              245250255                                                                      ThrTyrAspAsnAsnCysArgLeuGluCysAlaSerIleSerSerSer                               260265 270                                                                     ProGlyValGluLeuLysHisGluGlyIleCysArgLysGluGluLys                               275280285290                                                                   LysLeuProLysArgSerValGlyLeuGluHisThr CysValCysPro                              295300305                                                                      TyrAsnTyrPheProValCysGlyThrAspGlyGluThrTyrProAsn                               310315 320                                                                     LeuCysAlaLeuGlnCysArgMetArgGluValProGlyLeuGluLeu                               325330335                                                                      LysHisThrGlyLysCysLeuProHisLeuAspPheProAspProVal                               34 0345350                                                                     (2) INFORMATION FOR SEQ ID NO: 18:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                      GluGlyGlyGluProCysAlaCysProHisAla                                              1 510                                                                          (2) INFORMATION FOR SEQ ID NO: 19:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 32 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                      GAAGGTGGTGAACCNTGYGCNTGYCCNCAYGC 32                                            (2) INFORMATION FOR SEQ ID NO: 20:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 45 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                      CGAGGGGGATGGTCGACGGAAGCGACCTTTTTTTTTTTTTT TTTT45                               (2) INFORMATION FOR SEQ ID NO: 21:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                      CGAGGGGGATGGTCGACGG 19                                                         (2) INFORMATION FOR SEQ ID NO: 22:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                      GATGGTCGACGGAAGCGACC 20                                                    

We claim:
 1. An isolated and purified protein with thrombin-inhibitory action from assassin bugs of the genus Rhodnius, having an amino-acid sequence indicated in SEQ ID NO:
 3. 2. An isolated and purified protein with thrombin-inhibitory action, containing one or more domains with the amino-acid sequence indicated in SEQ ID NO:
 3. 3. A pharmaceutical composition comprising the protein defined in claim 1 and another anticoagulant factor.
 4. A method of treating a host in need of thrombin-inhibitory activity which comprises administering to said host an effective amount of the protein defined in claim
 1. 5. The method of claim 4, wherein the host is in need of treatment for a thrombosis. 